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W. D. Chamberlain, J. T. Rosenbaum, E. J. Lee; In vivo Imaging of Murine Corneal Nerve Response to Injury. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2502.
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To investigate and compare in vivo corneal nerve responses to different types of injury: chemical burn and surgical incision.
Mice which express yellow fluorescent protein (YFP) under the thy1 promoter were used to visualize corneal nerves. Nerve architecture was documented in vivo with wide-field fluorescence microscopy before (baseline), and up to 56 d after injury by silver nitrate application or partial thickness surgical blade incision. Some corneas were also removed and fixed for wholemount examination by confocal microscopy.
Uninjured corneas possessed fine YFP+ nerves in the sub-basal epithelium and larger caliber nerve fibers in the anterior stroma. Unexpectedly, polymorphic, nucleated YFP+ cells were observed in the limbus (sub-epithelial layer), but were absent from the cornea. One hour after injury nerve fibers in the immediate zone of injury lost fluorescence. Decline in fluorescence followed two patterns: 1) transient loss with restoration of nerve fibers after 7 days, or 2) extended loss with apparent corneal nerve sprouting from these areas of disruption starting after 21 days. In the latter, regenerated nerves did not resemble nerve architecture at baseline. Accumulation of fluorescence was also noted after injury and had two patterns: 1) beading or clumping of YFP near damaged nerve endings and 2) YFP+ nucleated cell accumulation in the cornea with enrichment in zones of damage. Some of these cells appeared to associate with nerves, and in vivo time-lapsed videomicroscopy suggested movement of these cells along nerve fibers.
We are able to monitor in vivo corneal nerve responses to acute injuries such as chemical burns and clear corneal incision similar to those used in ophthalmic surgery. Loss of fluorescence in corneal nerves after injury suggests transient changes in axoplasmic flow as well as long-term alterations in axonal structure. Accumulation and apparent migration of YFP+ nucleated cells may represent scavenger response of inflammatory cells with acquired fluorescence or thy1-expressing cells responding to corneal injury. Regeneration of nerve fibers was slow in all cases and did not restore baseline nerve architecture within the time period studied. Here we demonstrate long-term changes in corneal nerve architecture after injury that likely reflect loss of corneal neuronal function. These changes may correlate with prolonged susceptibility to ocular surface disease after initial healing response.
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